ACS Chem Neurosci. 2018 Aug 15;9(8):2025-2040. doi: 10.1021/acschemneuro.7b00450. Epub 2018 Feb 13.
Neuropeptides can have significant effects on neurons and synapses, but among the ∼250 predicted peptides in nematodes, few have been characterized functionally. Here, we report new neuropeptides in the 4 RME nerve ring motorneurons of the nematode Ascaris suum. These GABAergic neurons are involved in three-dimensional head movement. Mass spectrometry (MS) of single dissected RMEs detected a total of 12 neuropeptides (encoded by five genes), nine of which are novel. None of these are expressed in the DI/VI inhibitory GABAergic motorneurons that synapse onto body wall muscle. Using peptide sequences obtained by tandem MS, we cloned the peptide-encoding transcripts and synthesized riboprobes for in situ hybridization (ISH). This complementary technique corroborated the results from single-cell MS, showing that the dissections were not contaminated with adhering tissue from other cells. We also synthesized a multiple antigenic peptide to raise a highly specific antibody against one of the endogenous peptides, which labeled the same cells detected by MS and ISH. Our results show that the RMEs can be divided into two subsets: RMED/V (expressing afp-2, afp-15, Asu-nlp-58, and high levels of afp-16) and RMEL/R (expressing afp-15 and low levels of afp-4 and afp-16). Almost all of these peptides are bioactive in A. suum.
神经肽对神经元和突触有显著影响，但在线虫中预测的约 250 种肽中，只有少数具有功能性特征。在这里，我们报告了线虫 Ascaris suum 的 4 个 RME 神经环运动神经元中的新神经肽。这些 GABA 能神经元参与三维头部运动。对单个分离的 RME 进行质谱 (MS) 分析总共检测到 12 种神经肽（由 5 个基因编码），其中 9 种是新的。这些神经肽都不在与体壁肌肉突触的 DI/VI 抑制性 GABA 能运动神经元中表达。使用串联 MS 获得的肽序列，我们克隆了编码转录本的肽，并合成了用于原位杂交 (ISH) 的核糖探针。这种互补技术证实了单细胞 MS 的结果，表明这些分离物没有与来自其他细胞的粘着组织混合。我们还合成了一种多抗原肽，以针对一种内源性肽产生高度特异性抗体，该抗体标记了 MS 和 ISH 检测到的相同细胞。我们的结果表明，RME 可分为两个亚群：RMED/V（表达 afp-2、afp-15、Asu-nlp-58 和高水平的 afp-16）和 RMEL/R（表达 afp-15 和低水平的 afp-4 和 afp-16）。几乎所有这些肽在 A. suum 中都具有生物活性。
